This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research;2) exposing undergraduates to research;and 3) strengthening the research environment in non-research intensive universities. The goal of this research is to elucidate the mechanism of polyhydroxyalkanoate inclusion biogenesis. Electron microscopy studies have been unable to resolve the structure of PHA inclusions and this has inhibited movement toward a cohesive model of inclusion biogenesis. Employing atomic force microscopy, we have determined that there are three layers of structure, an outer envelope that is the thickness of a membrane bilayer, a middle network layer, and an underlying crystalline lamellar layer. Genetic studies have indicated that the middle network is comprised at least partially of PhaP and that PhaP is likely to be translocated to the periplasm. Thus, it would appear that inclusion biogenesis may occur by movement of protein and/or proteins to the periplasm and budding through the cytoplasmic membrane into the cytoplasm, facilitating the acquisition of the cytoplasmic membrane as an envelope. The goal of this research is to prove or disprove this supposition. The specific aims of the research are: 1) definitively prove periplasmic localization of PhaP via fluorescence localization and Western blot analyses of subcellular fractions, 2) demonstrate that the inclusion envelope is derived from the cytoplasmic membrane by proteomic analysis, and 3) characterize proteins that bind transiently and permanently to PhaP in hopes of elucidating the mechanism of inclusion biogenesis. Ultimately, the goal of the research is to enlarge our knowledge of inclusion biogenesis to the point that this process can be controlled and utilized for medical applications. For instance, it could be envisioned that instead of polymer being inserted into the inclusion, bioactive compounds could be inserted, making the inclusion into a drug delivery vehicle.